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CA1 Contributes to Microcalcification and Tumourigenesis in Breast Cancer Yabing Zheng1, Bing Xu1, Yan Zhao1,Hegu1, Chang Li2, Yao Wang1 and Xiaotian Chang1*

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CA1 Contributes to Microcalcification and Tumourigenesis in Breast Cancer Yabing Zheng1, Bing Xu1, Yan Zhao1,Hegu1, Chang Li2, Yao Wang1 and Xiaotian Chang1* Zheng et al. BMC Cancer (2015) 15:679 DOI 10.1186/s12885-015-1707-x RESEARCH ARTICLE Open Access CA1 contributes to microcalcification and tumourigenesis in breast cancer Yabing Zheng1, Bing Xu1, Yan Zhao1,HeGu1, Chang Li2, Yao Wang1 and Xiaotian Chang1* Abstract Background: Although mammary microcalcification is frequently observed and has been associated with poor survival in patients with breast cancer, the genesis of calcification remains unclear. Carbonic anhydrase I (CA1) has been shown to promote calcification by catalysing the hydration of CO2. This study aimed to determine whether CA1 was correlated with microcalcification and with other processes that are involved in breast cancer tumourigenesis. Methods: CA1 expression in breast cancer tissues and blood samples was detected using western blotting, real-time PCR, immunohistochemistry and ELISA. Calcification was induced in the cultured 4T1 cell line originating from mouse breast tumours, using ascorbic acid and β-glycerophosphate. Acetazolamide, a chemical inhibitor of CA1, was also added to the culture to determine the role of CA1 in calcification. The MCF-7 human breast cancer cell line was treated with anti-CA1 siRNA and was assessed using a CCK-8 cell proliferation assay, an annexin V cell apoptosis assay, transwell migration assay and a human breast cancer PCR array. The tag SNP rs725605, which is located in the CA1 locus, was genotyped using TaqMan® genotyping. Results: Increased CA1 expression was detected in samples of breast carcinoma tissues and blood obtained from patients with breast cancer. A total of 15.3 % of these blood samples exhibited a 2.1-fold or higher level of CA1 expression, compared to the average level of CA1 expression in samples from healthy controls. Following the induction of calcification of 4T1 cells, both the number of calcium-rich deposits and the expression of CA1 increased, whereas the calcification and CA1 expression were significantly supressed in the presence of acetazolamide. Increased migration and apoptosis were observed in MCF-7 cells that were treated with anti-CA1 siRNA. The PCR array detected up-regulation of the androgen receptor (AR) and down-regulation of X-box binding protein 1 (XBP1) in the treated MCF-7 cells. Significant differences in the allele and genotype frequencies of rs725605 were detected in the cohort of patients with breast cancer but not in other tumours. Conclusion: The results of this study suggested that CA1 is a potential oncogene and that it contributes to abnormal cell calcification, apoptosis and migration in breast cancer. Keywords: Breast cancer, Microcalcification, Tumourigenesis, Carbonic anhydrase I (CA1), Androgen receptor (AR), X-box binding protein 1 (XBP1) Background mammary calcifications: calcium oxalate and hydroxy- Mammary microcalcification is frequently associated apatite [2, 3]. Hydroxyapatite is also a well-documented with poor survival, and it occurs in 30 to 50 % of breast component of bone, and the deposition of it in bone tis- cancer patients [1]. Although the diagnostic value of sue requires the coordinated expression of several bone microcalcification in patients with breast cancer is of matrix proteins, which are synthesised by cells of osteo- great importance, the genesis of this calcification blastic lineage [4]. There has been evidence indicating remains unclear. There are two distinct forms of that calcium carbonate is involved in initial bone forma- tion, although mineral deposits are typically comprised – * Correspondence: [email protected] of calcium phosphate and not calcium carbonate [5 7]. 1Medical Research Center of Shandong Provincial Qianfoshan Hospital, CA1 (carbonic anhydrase 1) is a member of the carbonic Shandong University, Jingshi Road 16766, Jinan, Shandong 250014, P. R. China anhydrase (CA) family, and it catalyses the reversible Full list of author information is available at the end of the article © 2015 Zheng et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zheng et al. BMC Cancer (2015) 15:679 Page 2 of 15 hydration and dehydration reactions of CO2/H2CO3 [8]. Methods In vitro assays have demonstrated that CA1 not only Tissue collection enhances hydration reactions but also promotes the for- In the present study, all of the solid tissue samples and mation of CaCO3 [9, 10]. In a recent study, we detected blood samples that were used were collected from increased expression of CA1 in Saos-2, a human osteo- patients of Shandong Provincial Qianfoshan Hospital sarcoma cell line, following the induction of calcification (Jinan, China). The tumour diagnoses were verified with ascorbic acid and β-glycerophosphate. Following using histological methods, and pathological categorisa- treatment with acetazolamide, an anti-CA1 drug, both tions were performed according to the World Health CA1 expression and the formation of mineralised nod- Organisation (WHO) classification system. All of the ules decreased [11]. Furthermore, overexpression of CA1 included patients signed informed consent forms, and was detected in the synovial tissues of patients with an- this study was approved by the ethics committee of kylosing spondylitis, a disease characterised by abnormal Shandong Provincial Qianfoshan Hospital (reference bone formation in the spinal and sacroiliac joints [12]. number 2013012). All research involving human subjects The gene that encodes CA1 is susceptible to ankylosing (including human material or human data) that is spondylitis [11], and transgenic mice with overexpres- reported in the manuscript was conducted in compli- sion of CA1 exhibited bone fusion in their paws and ance with the Helsinki Declaration. spines [13]. These results suggest that CA1 might play essential roles in bio-mineralisation and new bone SNP selection and Taqman genotyping formation. The tag SNP rs725605 was identified by searching the In the present study, we hypothesised that the up- HapMap database. This tag SNP was selected on the regulation of CA1 in breast tumours stimulates calcium basis of linkage disequilibrium patterns that were ob- precipitation, similar to what occurs in bone tissue. To served in samples obtained from the Han Chinese popu- test this hypothesis, calcification was induced in 4T1 lation of Beijing, who were genotyped as part of the cells, which originated from a murine mammary adenocar- International HapMap Project. Illumina microarray and cinoma. Cox et al. recently reported a novel in vitro model Taqman genotyping assays confirmed the location of tag of mammary mineralisation using 4T1 cells [14, 15]. Using SNP rs725605 in the CA1 locus [11]. this model, we investigated the involvement of CA1 in To determine the potential associations between this bio-mineralisation and calcification. Additionally, the tag SNP and various cancer risks, genotyping was effects of CA1 on cell proliferation and cell apoptosis performed using TaqMan® technology in cohorts of were investigated in anti-CA1 siRNA-treated MCF-7 patients who had breast cancer (n = 285, 285 women, cells that originated from human breast cancer, and the mean age = 47.65 years old), colon cancer (n = 145, 55 pathogenic pathway was analysed using a PCR array women, mean age = 54.13 years old), oesophageal cancer that contained breast cancer genes. We also used a Taq- (n = 285, 40 women, mean age = 61.20 years old), cer- man genotyping method to determine the correlations vical cancer (n = 190, 190 women, mean age = 52.75 years between common polymorphisms in the CA1 encoding old), liver cancer (n = 190, 42 women, mean age = gene and breast cancer. In a previous study, we genotyped 54.05 years old), lung cancer (n = 190, 56 women, mean 13 different tag SNPs in the CA1 gene to determine age = 58.17 years old), gastric cancer (n =285,71women, whether a potential association existed between the CA1 mean age = 56.83 years old) or rectal cancer (n =137,50 gene and ankylosing spondylitis, using a custom-designed women, mean age = 54.61 years old), as well as in healthy Illumina 96-SNP VeraCode microarray (Illumina). We controls (n = 285, 71 women, mean age = 38.42 years old). subsequently performed Taqman genotyping of the tag Blood samples were collected and stored in Monovette SNPs that exhibited significant associations with the tubes containing 3.8 % sodium citrate. disease in a large cohort of the patients. Both microarray Genomic DNA was extracted from whole blood samples and Taqman genotyping demonstrated that the allele and with an Omega E-Z 96 Blood DNA kit (Omega, USA) gene frequencies of rs725605 were statistically significantly according to the manufacturer’s protocol. Genotyping associated with patients who had ankylosing spondylitis assays were run on a ViiA 7 DX (Life Technology) and [11]. At that time, we tested five SNPs by Taqman geno- were evaluated according to the manufacturer’sinstruc- typing, and only rs725605 produced favourable results. tions. Each reaction was performed in a total volume of Thus, we continued to genotype this tag SNP to deter- 10 μl using the following amplification protocol: denatur- mine whether the CA1 gene had possible associations ation at 95 °C for 10 min, followed by 50 cycles of with the various types of tumours that were examined in denaturation at 95 °C for 15 s and finishing with annealing the current study. Furthermore, we examined the CA1 and extension at 60 °C for 1 min. The genotype of each expression in breast tumour tissues and blood samples sample was determined by measuring allele-specific fluor- from patients. escence using Taqman Genotyper software, version 1.2 Zheng et al. BMC Cancer (2015) 15:679 Page 3 of 15 (Life Technology).
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